The present invention relates to a process for fermenting carbohydrates using bacteria of the genus Zymomonas initially inoculated into yeast-conditioned media.
Ethanol production has traditionally been carried out in a two-stage, batch process employing yeast. The first stage of this process, in which the yeast are grown under aerobic conditions, is referred to as the "growth stage". The second stage entails the production, by fermentation, of ethanol under anaerobic conditions, i.e., in the presence of only small amounts, if any, of oxygen. Addition of air or oxygen is required if the yeast is to be propagated during the ethanol-producing second stage. In particular, oxygen is required if the efficiency of the total process is to be increased by the occasional recycling of yeast cells, for example, via sedimentation or centrifugation.
One disadvantage of the traditional process of yeast fermentation is its long fermentation time. "Fermentation time" is the period of time required for complete conversion of substrate to end products, or for the biocatalyst (the ethanol producing organisms) to reach the stage of maximum yield of end-products. At a preferred optimal temperature of between 30.degree. and 40.degree. C., 30- to 70-hour fermentation times have been necessary to obtain 9% to 11% (v/v) ethanol when yeast cells are used for conversion of glucose to ethanol. The rate-limiting factors in this process are the rates of glucose uptake and ethanol production by individual yeast cells. These rates are stringently regulated by cellular enzyme control systems.
One method devised to increase the yield of ethanol is to use a larger inoculum of yeast cells, i.e., a larger biomass density. However, even when using a cell density of 5-10 million cells per milliliter (mL), this method still requires 30-50 hours of fermentation time. Reduction in fermentation time to the range of 10-30 hours has been achieved by using 30-100 times higher biomass density in a two-stage batch process. This higher biomass density is achieved by recycling cells previously used in another fermentation. However, the higher biomass density requires an adequate supply of nutrients, supplies of which are often limited.
Another disadvantage of the yeast fermentation process, especially when cost is a major concern, is the diversion of fermentable substrate to the production of by-products such as glycerol, amylalcohol and other fusel oils. As a result, the efficiency of conversion of substrate to end-product, e.g., from glucose to ethanol, is lower than desirable, i.e., only between 85 and 91%.
Efforts to increase the yield of ethanol from sugar fermentation has also been limited by the sensitivity of yeast cells to high concentrations of both the sugar substrate ("substrate inhibition") and the ethanol end-product ("end-product inhibition"). Substrate inhibition results from an osmotic effect of the substrate on the cells, and is reflected in reduced water activity inside the cell, plasmolysis, and decreased viability. End-product inhibition results from high solubility of cell membranes in ethanol and feedback inhibition. In either case, there is inhibition of cell growth or fermentation.
To overcome the drawbacks of yeast-based fermentation, attempts have been made to use Zymomonas bacteria instead of yeast. Zymomonads, such as Z. mobilis, are a very limited class of facultative anaerobic bacteria that metabolize glucose via the Entner-Doudoroff pathway, usually found in strictly aerobic microorganisms. Strains of Z. mobilis have been shown to have rates of glucose uptake and ethanol production, respectively, that are several times higher than yeast, as well as higher ethanol-yield values, reflecting the ability of the strains to tolerate high concentrations of both sugar and ethanol.
Notwithstanding the potential advantages of Zymomonas bacteria compared to yeast, the development of practical fermentation systems employing the bacteria has lagged, at least in part due to a serious contamination problem associated with the use of Zymomonas in high-sugar fermentation environments. Gram-negative Zymomonas cells growing under commercial plant conditions are often overwhelmed by competition from contaminating microflora, principally gram-positive bacteria, before the fermentative bacteria can become established, effectively precluding the increase in zymomonad biomass that is necessary to accomplish industrial-scale fermentation, typically requiring volumes greater than 1000 gallons.
In the face of problems with both yeast-based and zymomonad-based fermentations, it has been proposed to use mixed cultures of yeast and Zymomonas cells under conditions that would permit both types of fermentative microorganisms to function in the same environment. Generally, a mixed-culture is prepared from yeast and bacteria which have initially been cultured separately until they reach a logarithmic growth phase, that are then mixed together in similar proportions. The yeast/bacterial mixture is then added to a substrate, such as sucrose, fructose, or glucose, that is fermentable by both the yeast and Zymomonas.
The conditions present in a mixed culture must be applicable to the reproduction and fermentation of both organisms; i.e., both yeast and Zymomonas. A mixed culture requires that the populations of the two organisms reach an equilibrium, and remain stable throughout the fermentation process.
It is very difficult to maintain culture conditions which allow for reproduction of both organisms and maximum fermentation by both organisms. In order to maintain conditions for the propagation of one organism, the other will suffer. For example, air is needed for yeast to propagate, however, air interferes with the fermentation capabilities of the Zymomonas. Anaerobic conditions, on the other hand, will promote ethanol production by both the yeast and Zymomonas; however, the yeast will not propagate, their population will diminish, and a stable population will not be maintained. High ethanol concentrations will also inhibit yeast, whereas Zymomonas have been reported by Burrill et al., Biotech Letters 5(6), 423-428 (1983), to tolerate up to 150 ml ethanol/l before being inhibited. Further, Zymomonas are also unable to utilize urea, a nutrient that is the most commonly accepted nutrient for ethanol production by yeasts in the ethanol industry today.
Mixed cultures are not suitable in many of the fermentation methods already in use in the ethanol industry. For example, mixed cultures of yeast and zymonads cannot be grown using continuous fermentation, such as the Vogelbusch technology, which is based on the maintenance of a steady state population of a particular organism by using the dilution rate of the media to maintain and equal the growth rate of the organism. Every organism has its own growth rate at any particular dilution rate which determines which organism will predominate. If a contaminant organism has a higher growth rate than the yeast, it will become the dominant organism; on the other hand, if the yeast has the higher growth rate, the yeast will predominate.
Other conditions, such as pH, temperature, antibiotics, and nutritional supplements, can also alter the growth rate of an organism, giving the opportunity for a new organism to take over. Under conditions such as these, it is virtually impossible for two organisms to remain co-dominant, as required for the successful functioning of a mixed culture.
Even where the two organisms in a mixed culture can be grown together successfully, the disadvantage remains with respect to production of ethanol by the yeast, i.e., the yeast will still produce fusel oils and the yield of ethanol from substrate will remain unchanged.
It is therefore an object of the present invention to provide a fermentation process that exploits the beneficial properties of Zymomonas while avoiding problems with contamination by other bacteria.
It is another object of the present invention to provide a fermentation system that can accommodate high-sugar feedstocks, affording high ethanol-yield values, without the necessity of maintaining mixed yeast-Zymomonas cultures.
It is still another object of the present invention to improve the efficiency of ethanol fermentation by eliminating simultaneous fusel oil production.
It is a further object of the present invention to minimize the time required for ethanol fermentation by Zymomonas.